In human airways local nucleotide release initiates paracrine and autocrine pathways that regulate mucus clearance by stimulating mucus secretion, ion transport, and ciliary beating. Although CFTR activity is clearly important for efficient mucociliary clearance, little is known about the mechanisms that transduce physical stresses into activation of CFTR. We hypothesize that following release of ATP onto the airway surface, ATP is metabolized to adenosine, which acts via A2B receptors to regulate CFTR function. We hypothesize that following release of ATP onto the airway surface, ATP is metabolized to adenosine, which acts via A2B receptors to regulate CFTR function. We will test this hypothesis in freshly excised tissues and in CalU3 cells, a serous cell model that expresses readily detectable CFTR function and protein. A2B receptors stimulate adenylate cyclase to increase intracellular cAMP, and we previously showed that cAMP dependent protein kinase A must be anchored at the apical membrane for efficient regulation of CFTR by adenosine. The balance between adenylate cyclase and phosphodiesterase controls the local concentration of cAMP, and protein kinase A activity, in subcellular compartments. Therefore, we hypothesize that specific isoforms of adenylate cyclase and phosphodiesterase are also compartmentalized at the apical membrane in close proximity with CFTR. We will test this hypothesis functionally in excised apical membrane patches from CalU3 cells and will determine the specific isoforms of each protein that are involved. We find that protein kinase C activity is required for protein kinase A to regulate CFTR. Therefore, we will test the hypothesis that PKC must be present at the apical membrane for efficient modulation of CFTR by adenosine, we will identify the specific PKC isozyme(s) involved. We will also identify proteins that target PKC to the apical cell surface in airway. The experiments will provide an in-depth understanding of a signal transduction pathway of a signal transduction pathway that regulates the local concentration of cAMP in the apical compartment of airway cells. Since cAMP is the dominant regulator of CFTR, the experiments will provide insights into the regulation of C1-transport in airway epithelial and will suggest how cAMP efficiency regulates other components of mucociliary clearance.